We investigate the molecular mechanisms that control the sorting of transmembrane proteins in the endosomal-lysosomal system. Sorting is mediated by interactions between signals in the cytosolic domains of the transmembrane proteins and adaptor protein associated with the cytosolic face of membranes. Two major types of sorting signals, referred to as tyrosine-based and dileucine-based, have been previously described. Work in this section has demonstrated that both types of signals are recognized with characteristic fine specificities by the adaptor protein (AP) complexes AP-1, AP-2, AP-3 and AP-4, or by the GGA adaptor proteins GGA1, GGA2 and GGA3. Mutations in AP-3 are the cause of the pigmentation and bleeding disorder, Hermansky-Pudlak syndrome type 2. Current work is aimed at elucidating the structure, regulation and physiological roles of the AP complexes and GGAs, and investigating the possibility that defects in these proteins underlie protein trafficking disorders. Over the past year, we have continued our studies on the structure and function of the GGA proteins and AP complexes. The GGAs function as Arf-dependent adaptors for the recruitment of clathrin to the TGN. In addition, they participate in the sorting of mannose 6-phosphate receptors (MPRs) and their cargo, the lysosomal hydrolases, from the TGN. We have identified a canonical peptide motif that mediates interaction of accessory proteins with the "ear" domain of the GGAs and the related domains of the gamma-adaptin subunit of AP-1. In collaboration with the group of James Hurley (NIDDK), we have solved the crystal structure of the GAE domain of human GGA3 in complex with a Phe-Gly-Pro-Leu-Val peptide, derived from the accessory protein Rabaptin-5, which conforms to the canonical peptide motif. We have also perfromed studies on the function of AP complexes. In particular, we succeeded in identifying the subunits of AP complexes that interact with dileucine-based signals from HIV-1 Nef and the lysosomal membrane protein Limp II. The characterization of the molecular machinery involved in protein sorting is important for the understanding of the pathogenesis of various metabolic and developmental disorders. An example of such a disorder is the Hermansky-Pudlak syndrome (HPS), a genetically heterogeneous heritable disease that affects lysosome-related organelles such as melanosomes and platelet dense bodies. We have previously demonstrated that mutations in the gene encoding the beta3A subunit of AP-3 are the cause of HPS type 2. Strikingly, mutations in at least three other genes in humans and 14 genes in mice cause a similar disorder. Most of the HPS genes identified to date by positional cloning encode proteins of unknown function and no recognizable homology to other proteins. To gain insight into the nature of this machinery, we have undertaken a biochemical characterization of the novel HPS gene products. We previously found that the protein products of the pallid, muted and cappuccino genes are the components of a novel complex named BLOC-1. This past year, we demonstrated that the product of the reduced pigmentation gene is a novel component of BLOC-1 and that the products of the pale ear and light ear genes are part of another complex, which we named BLOC-3. This complex is found both in the cytosol and peripherally associated with membranes. Its properties are consistent with it being a component of the molecular machinery for biogenesis of lysosome-related organelles. Ongoing studies on BLOC-1 and BLOC-3 are likely to provide additional insights into the pathogenesis of HPS.